The long-term objectives of this project are to clarify mechanisms that regulate proliferation of adrenal chromaffin cells, to identify regulatory abnormalities that occur during development and progression of chromaffin cell tumors (pheochromocytomas) and to relate those abnormalities to genetic defects that predispose to tumor formation. The present proposal is based on three principal findings from the past funding period: 1 .Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN), which activate the tyrosine kinase, ret, are mitogenic for adult rat chromaffin cells in vitro. 2.Depolarization and activation of protein kinase C (PKC), which mimic trans-synaptic stimulation, potentiate mitogenicity of those neurotrophic factors by increasing the proportion of cells that respond. 3.Corticosterone, the major adrenal cortical steroid in the rat, is necessary for substantial expansion of chromaffin cell populations stimulated to proliferate. These findings suggest that neurally derived signals in vivo might stimulate chromaffin cell proliferation by increasing the activation of ret, and that corticosteroids play a cooperative role by promoting survival of cells that proliferate. The effects of depolarization, which opens voltage-gated Ca++ channels, and PKC, which is functionally regulated by cytosolic Ca++ suggest that optimal mitogenesis by ret-activating ligands requires an integrated signal involving neurally mediated increases in PKC activity and intracellular Ca++ The proposed studies will dissect the mechanisms that mediate mitogenesis by GDNF and NTN and will determine how those mechanisms are affected by corticosterone. Four principal hypotheses will be tested: 1. That recruitment of chromaffin cells to proliferate results from increased expression of GDNF and NTN receptors and ret. 2. That mitogenesis by GDNF or NTN utilizes specific subtypes of Ca++ channels that are selectively activated or up-regulated under different conditions to achieve optimal mitogenic signaling. 3. That mitogenesis by NTN and GDNF is ret-dependent, and a function of specific pathways activated by those factors is to further increase ret expression. 4. That corticosterone promotes survival of proliferating chromaffin cells that would otherwise die, and the protective mechanisms involve components of ret signaling pathways known to be neuroprotective. Ret is expressed in human pheochromocytomas. In addition, ret mutations causing constitutive activation or altered substrate affinity lead to the development of pheochromocytomas in Multiple Endocrine Neoplasia syndromes 2A and 2B. Our findings to date establish the utility of the rat chromaffin cell model for studies of ret signaling. The proposed studies will help to elucidate mechanisms that may be involved in the development and progression of pheochromocytomas.